1. Field of the Invention
This invention provides polynucleotide constructs encoding constitutively active membrane-targeted PI 3-kinase mutants, methods for making polynucleotide constructs, an in vivo method for screening for inhibitors of PI 3-kinase using the constructs, use of the polynucleotide constructs to prevent cell death, or to restore insulin responsiveness in type II diabetes, use of the polynucleotide constructs to express PI 3-kinase mutants that generate 3xe2x80x2 phosphorylated inositol phospholipids, and use of these phospholipids to prevent cell death.
2. Background of the Invention
Phosphotidylinositol (PI) 3-kinase, both a phospholipid kinase, and a protein serine/threonine kinase, is implicated in certain oncogenic or mitogenic responses. See Carpenter et al., Mol. Cell. Biol. 13:1657-1665 (1993), Cantley et al., Cell 64:281-302 (1991), Escobedo and Williams, Nature 335:85-87 (1988), and Fantl et al, Cell 69: 413423 (1992). It is an intracellular heterodimer consisting of an 85-kDa regulatory subunit (p85), and a 110-kDa catalytic subunit (p110) that is stimulated by growth factors. See Whitman et al., Nature 332:644-646 (1988). The p85 subunit contains several domains and links the catalytic subunit to activated growth factor receptors. The cDNA for the p110 subunit has recently been cloned and expressed in insect and mammalian systems as described in Hiles et al., Cell 70:419-429 (1992). The general structure and function of PI 3-kinase, including analysis of the structure and function of its subunits p85 and p110, are described in Klippel et al., Mol. Cell. Biol. 13:5560-5566 (1993), and in Klippel et al., Mol. Cell. Biol. 14:2675-2685 (1994).
The p85 subunit of PI 3-kinase has several domains, including a 200 amino acid region of p85 located between the two SH2 domains. This domain, called the inter-SH2 or iSH2 domain, has been found sufficient to promote interaction with p110 in vivo with activity comparable to that of full-length p85. See Klippel et al., Mol. Cell. Biol. 13:5560-5566 (1993). Additionally, a complex between a 102 amino acid segment of p85 and the p110 subunit has been found to be catalytically active, as described in and Klippel et al., Mol. Cell. Biol. 14:2675-2685 (1994).
Previously, studies to elucidate the of PI 3-kinase activation have been conducted by constructing receptor mutants to alter the signal transduction of PI 3-kinase, or by constructing mutant oncogenes to study a PI 3-kinase inducible oncogenic response. It would be advantageous to study effects of PI 3-kinase activation directly, without growth factor activation, so as to identify the role of PI 3-kinase in oncogenesis, mitogenesis, and other tyrosine kinase and PI related functions. Methods and compositions derived from such knowledge and use of PI 3-kinase to control oncogenesis or mitogenesis, would be advantageous in the treatment of cancer. In addition, it would be advantageous to develop methods and compositions for such applications as preventing cell death or treating type II diabetes.
In one aspect, the invention provides polynucleotide sequences comprising the p110 subunit of PI 3-kinase polynucleotide attached to a cell membrane targeting sequence. More specifically, the invention provides a polynucleotide sequence comprising a first nucleotide sequence encoding the p110 subunit of PI 3-kinase protein, or a derivative or mutant of this sequence having a single or multiple nucleotide substitution, deletion or addition, this derivative or mutant having p110 catalytic activity, and a second nucleotide sequence encoding a cell membrane targeting sequence, this second nucleotide sequence being attached to the first nucleotide sequence at the latter""s 5xe2x80x2 or 3xe2x80x2 end. Further, the polynucleotide sequence of the invention can be structured so that the first nucleotide sequence also includes a nucleotide sequence encoding the p85 subunit of PI 3-kinase or a fragment of the p85 subunit, for example the iSH2 domain of the p85 subunit, capable of binding the p110 subunit. The cell membrane targeting sequence is a nucleotide sequence encoding a myristoylation, or a palmitoylation and farnesylation amino acid sequence.
Other aspects of the invention include methods of screening for inhibitors of PI 3-kinase, methods of making 3xe2x80x2 phosphorylated inositol phospholipids, and the 3xe2x80x2 phosphorylated inositol phospholipid produced thereby, and methods for activating enzyme effectors of PI 3-kinase having a pleckstrin homology domain.
Therapeutic aspects of the invention include methods of reducing cell death due to trauma, by administering to the cell a viral or non-viral vector including a polynucleotide sequence of the invention, or by administering a 3xe2x80x2 phosphorylated inositol phospholipid to the cell. Another aspect of the invention is a method of promoting activation of an insulin signaling pathway by contacting a cell characterized by insulin resistance with a vector having a polynucleotide sequence of the invention.